CAJ | 학술논문

目的：构建高滴度大鼠N1ICD慢病毒过表达载体（LV-N1ICD）及N1ICD慢病毒干扰载体（LV-N1ICD-shRNA）。方法以大鼠cDNA文库为模板，PCR法扩增N1ICD，通过定向克隆构建pGC-FU-N1ICD-3Flag穿梭质粒；设计4对N1ICD-shRNA寡核苷酸序列，以构建GVC112-N1ICD-shRNA干扰质粒，将pGC-FU-N1ICD-3Flag和GVC112-N1ICD-shRNA共转293T细胞，检测Flag的表达，筛选理想的GVC112-N1ICD-shRNA干扰质粒。将pGC-FU-N1ICD-3Flag或GVC112-N1ICD-shRNA与pHelper 1.0、pHelper 2.0共转293T细胞，以包装LV-N1ICD和LV-N1ICD-shRNA，分别利用Real-time PCR、药物筛选法进行病毒滴度测定。LV-N1ICD及LV-N1ICD-shRNA分别感染H9c2心肌细胞，利用CCK-8检测细胞活力。结果 pGC-FU-N1ICD-3Flag和GVC112-N1ICD-shRNA质粒经PCR、基因测序及Western-blotting验证构建成功，与pHelper 1.0、pHelper 2.0共转293T细胞后，取上清浓缩，分别获得高滴度LV-N1ICD和LV-N1ICD-shRNA。LV-N1ICD可明显提高心肌细胞活力，LV-N1ICD-shRNA可降低心肌细胞活力。结论 LV-N1ICD和LV-N1ICD-shRNA包装成功，具有Notch1信号通路生物学功能。
목적：구건고적도대서N1ICD만병독과표체재체（LV-N1ICD）급N1ICD만병독간우재체（LV-N1ICD-shRNA）。방법이대서cDNA문고위모판，PCR법확증N1ICD，통과정향극륭구건pGC-FU-N1ICD-3Flag천사질립；설계4대N1ICD-shRNA과핵감산서렬，이구건GVC112-N1ICD-shRNA간우질립，장pGC-FU-N1ICD-3Flag화GVC112-N1ICD-shRNA공전293T세포，검측Flag적표체，사선이상적GVC112-N1ICD-shRNA간우질립。장pGC-FU-N1ICD-3Flag혹GVC112-N1ICD-shRNA여pHelper 1.0、pHelper 2.0공전293T세포，이포장LV-N1ICD화LV-N1ICD-shRNA，분별이용Real-time PCR、약물사선법진행병독적도측정。LV-N1ICD급LV-N1ICD-shRNA분별감염H9c2심기세포，이용CCK-8검측세포활력。결과 pGC-FU-N1ICD-3Flag화GVC112-N1ICD-shRNA질립경PCR、기인측서급Western-blotting험증구건성공，여pHelper 1.0、pHelper 2.0공전293T세포후，취상청농축，분별획득고적도LV-N1ICD화LV-N1ICD-shRNA。LV-N1ICD가명현제고심기세포활력，LV-N1ICD-shRNA가강저심기세포활력。결론 LV-N1ICD화LV-N1ICD-shRNA포장성공，구유Notch1신호통로생물학공능。
Objective To construct rat N1ICD lentiviral over-expression vector (LV-N1ICD) and N1ICD lentivirus interference vector (LV-N1ICD-shRNA. Methods With the rat cDNA as a template, the N1ICD fragment was amplified by PCR to construct pGC-FU-N1ICD-3Flag shuttle plasmid by directly clone. Four pairs of N1ICD-shRNA oligonucleotide sequences were syn-thesized to construct the GVC112-N1ICD-shRNA interference plasmid. pGC-FU-N1ICD-3Flag and GVC112-N1ICD-shRNA plasmids were co-transfected into 293T cells to screen for the best interference plasmid in the 4 GVC112-N1ICD-shRNA plasmids by detecting Flag expression. pGC-FU-N1ICD-3Flag or GVC112-N1ICD-shRNA plasmid along with with pHelper 1.0 and pHelper 2.0 plasmids were co-transfect into 293T cells to package LV-N1ICD and LV-N1ICD-shRNA, and the virus titer was determined by real-time PCR and drug screening method, respectively. H9c2 cells infected with LV-N1ICD and LV-N1ICD-shRNA respectively were assessed for cell viability using CCK-8 assay. Results pGC-FU-N1ICD-3Flag and GVC112-N1ICD-shRNA plasmid were verified by PCR, gene sequencing and Western blotting. Co-transfection of the plasmids with pHelper 1.0, and pHelper 2.0 plasmids into 293T cells obtained high-titer LV-N1ICD and LV-N1ICD-shRNA. LV-N1ICD was capable of promoting the cell viability and LV-N1ICD-shRNA produced an opposite effect. Conclusion The vectors LV-N1ICD and LV-N1ICD-shRNA have been successfully constructed and packaged, which have the biological functions of Notch1 signaling.